Methods for the detection of anti-drug antibodies against factor xi and/or factor xia antibodies

Abstract

This disclosure relates to methods for detection and measurement of anti-drug antibodies (ADAs) against Factor XI and/or Factor XIa therapeutic antibodies, e.g., in a subject being treated with said Factor XI and/or Factor XIa therapeutic antibodies.

1 - 56 . (canceled) 
     
     
         57 . A method of detecting an anti-drug antibody (ADA) against an anti-Factor XI and/or anti-Factor XIa (anti-FXI/FXIa) antibody or antigen-binding fragment thereof, wherein the method comprises:
 (a) incubating a sample with an acid to dissociate anti-FXI/FXIa antibody-antigen complexes and/or dissociate anti-FXI/FXIa antibody-ADA complexes present in the sample to create an acid digest;   (b) incubating the acid digest on a plate coated with the anti-Factor XI and/or anti-Factor XIa antibody or antigen-binding fragment thereof;   (c) neutralizing the acid digest; and   (d) detecting the presence of the ADA using a ruthenylated detector cocktail.   
     
     
         58 . The method of  claim 57 , wherein the sample is selected from the group consisting of human blood, plasma, or serum. 
     
     
         59 . The method of  claim 57 , wherein the method further comprises:
 (a) an initial step of preparing the sample; and/or   (b) a washing step after the incubating.   
     
     
         60 . The method of  claim 57 , wherein the acid is selected from the group consisting of acetic acid, citric acid, phosphoric acid, and mixtures thereof. 
     
     
         61 . The method of  claim 60 , wherein the acid is acetic acid. 
     
     
         62 . The method of  claim 57 , wherein neutralizing uses a base selected from the group consisting of Tris, Phosphate, HEPES, triethanolamine, and mixtures thereof. 
     
     
         63 . The method of  claim 62 , wherein the base is Tris. 
     
     
         64 . The method of  claim 62 , wherein:
 (a) the base is at a pH of about 8.0; and/or   (b) the acid is at a concentration of about 300 mM.   
     
     
         65 . The method of  claim 57 , wherein the ruthenylated detector cocktail comprises a detector antibody. 
     
     
         66 . The method of  claim 65 , wherein the detector antibody is selected from the group consisting of an anti-human IgG, an anti-human IgM, an anti-human IgE, an anti-rabbit Ig, and any combination thereof. 
     
     
         67 . The method of  claim 57 , wherein the plate is coated with streptavidin. 
     
     
         68 . The method of  claim 57 , wherein the anti-FXI/FXIa antibody or antigen-binding fragment is coated on the plate in a buffer at a concentration selected from the group consisting of about 0.1 μg/mL, about 0.25 μg/mL, about 0.5 μg/mL, about 0.75 μg/mL, and about 1 μg/mL. 
     
     
         69 . The method of  claim 68 , wherein the anti-anti-FXI/FXIa antibody or antigen-binding fragment is coated on the plate in a buffer at a concentration of about 0.25 μg/mL. 
     
     
         70 . The method of  claim 57 , wherein the anti-FXI/FXIa antibody or antigen-binding fragment comprises:
 i. a heavy chain variable region CDR1 of SEQ ID NO: 23; a heavy chain variable region CDR2 of SEQ ID NO: 24; a heavy chain variable region CDR3 of SEQ ID NO: 25; a light chain variable region CDR1 of SEQ ID NO: 33; a light chain variable region CDR2 of SEQ ID NO: 34;and a light chain variable region CDR3 of SEQ ID NO: 35 ;   ii. a heavy chain variable region CDR1 of SEQ ID NO: 26; a heavy chain variable region CDR2 of SEQ ID NO: 27; a heavy chain variable region CDR3 of SEQ ID NO: 28; a light chain variable region CDR1 of SEQ ID NO: 36; a light chain variable region CDR2 of SEQ ID NO: 37;and a light chain variable region CDR3 of SEQ ID NO: 38;   iii. a heavy chain variable region CDR1 of SEQ ID NO: 43; a heavy chain variable region CDR2 of SEQ ID NO: 44; a heavy chain variable region CDR3 of SEQ ID NO: 45; a light chain variable region CDR1 of SEQ ID NO: 47; a light chain variable region CDR2 of SEQ ID NO: 37;and a light chain variable region CDR3 of SEQ ID NO: 15; or   iv. a heavy chain variable region CDR1 of SEQ ID NO: 46; a heavy chain variable region CDR2 of SEQ ID NO: 4; a heavy chain variable region CDR3 of SEQ ID NO: 5; a light chain variable region CDR1 of SEQ ID NO: 33; a light chain variable region CDR2 of SEQ ID NO: 14;and a light chain variable region CDR3 of SEQ ID NO: 15.   
     
     
         71 . The method of  claim 57 , wherein the anti-FXI/FXIa antibody or antigen-binding fragment comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 29, and a VH with 90% identity thereto; and a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 39, and a VL with 90% identity thereto. 
     
     
         72 . The method of  claim 57 , wherein the anti-FXI/FXIa antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 29; and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 and 39. 
     
     
         73 . The method of  claim 57 , wherein the anti-FXI/FXIa antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NOs: 31, 11, or a heavy chain with 90% identity thereto; and a light chain comprising an amino acid sequence of SEQ ID NOs: 41, 21, or a light chain with 90% identity thereto. 
     
     
         74 . The method of  claim 57 , wherein the anti-FXI/FXIa antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 31 and a light chain comprising an amino acid sequence of SEQ ID NO: 41. 
     
     
         75 . The method of  claim 57 , wherein the anti-FXI/FXIa antibody is a human monoclonal antibody. 
     
     
         76 . The method of  claim 75 , wherein the anti-FXI/FXIa antibody is a human IgG1isotype. 
     
     
         77 . The method of  claim 76 , wherein the anti-FXI/FXIa antibody comprises an antibody Fc domain comprising D265A and P329A substitutions.